High-molecular-weight adiponectin measurement method
a high-molecular weight, adiponectin technology, applied in the direction of instruments, drug compositions, metabolic disorders, etc., can solve the problems of inability to obtain precise results for assaying an hmw fraction, and inability to selectively determine the level of adiponectin multimers
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example 1
Digestion Specificity to Adiponectin Multimers in Mouse Serum
[0040]α-Chymotrypsin (Type I-S; No. C7762, product of Sigma-Aldrich) was dissolved in 50 mM Tris-HCl buffer (pH: 8.0), to thereby prepare an enzyme solution having a concentration of 10 u / mL, and the enzyme solution (100 μL) was added to mouse serum (10 μL). The mixture was incubated at 37° C. for 20 minutes. To the reaction mixture, BSA-PBST (20mM phosphate buffer containing 1% bovine serum albumin and 0.05% Tween 20, pH: 7.2) (400 μL) was added. The entire mixture was subjected to gel filtration chromatography by means of Superdex 200 (product of GE Healthcare Bio-Sciences). PBS (20 mM phosphate buffer, pH: 7.2) was used as an eluent, and fractions (1 mL each) were collected. As controls, fractions obtained through gel filtration chromatography under the same conditions except that no chymotrypsin was added, were used.
[0041]Detection of adiponectin in each fraction was performed in the following manner. An anti-mouse adi...
example 2
Digestion Specificity to Adiponectin Multimers in Rat Serum
[0043]α-Chymotrypsin- (Type I-S; No. C76-2, product of Sigma-Aldrich) was dissolved in 50 mM Tris-HCl buffer (pH: 8.0), to thereby prepare an enzyme solution having a concentration of 100 u / mL, and the enzyme solution (100μL) was added to rat serum (50 μL). The mixture was incubated at 37° C. for 20 minutes. To the reaction mixture, BSA-PBST (400 μL) was added. The entire mixture was subjected to gel filtration chromatography by means of Superdex 200 (product of GE Healthcare Bio-Sciences). PBS was used as an eluent, and fractions (1 mL each) were collected. As controls, fractions obtained through gel filtration chromatography under the same conditions except that no chymotrypsin was added, were used.
[0044]Detection of adiponectin in each fraction was performed in the following manner. A goat anti-rat adiponectin polyclonal antibody (product of R&D Systems) was diluted with PBS to a concentration of 0.5 μg / mL, and added to a...
referential example 1
Digestion Specificity to Adiponectin Multimers when Trypsin was Used
[0046]Trypsin (No. T1426, product of Sigma-Aldrich) was dissolved in 50 mM Tris-HCl buffer (pH: 8.0), to thereby prepare enzyme solutions having a concentration of 0 to 50 u / mL, and each enzyme solution (100 μL) was added to mouse serum, rat serum, and human serum (10 μL each). Each mixture was incubated at 37° C. for 20 minutes. To the reaction mixture, BSA-PBST (400 μL) was added. A portion of the mixture was separated through native polyacrylamide gel electrophoresis (native-PAGE) and transferred to PVDF membrane through semi-dry blotting, followed by immunostaining. In a specific procedure, the transferred membrane was blocked with BSA-PBST and washed with PBST. Then, a goat anti-mouse adiponectin polyclonal antibody (product of R&D systems) (0.1 μg / mL) was reacted with the membrane at room temperature for one hour. The membrane was sufficiently washed with PBST, and color development was performed by use of Vec...
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